Melanoma remains one of the deadliest of human cancers with no effective method for treating the disseminated disease. While targeted therapies have shown some efficacy, they have thus far proven to be noncurative, which points to the need for other forms of systemic therapy. This proposal is part of a long-term effort to develop methods for systemic therapy of melanoma. We have recently shown that administration of lonidamine (LND, 100 mg/kg) substantially enhances the activity of melphalan and have now extended this finding to doxorubicin treatment. As a key step towards eventual clinical translation, we will now examine the detailed mechanism of LND activity. We and others have shown that LND produces tumor specific intracellular acidification and a substantial decrease in tumor energy status (NTP/Pi). A number of mechanisms have been proposed for LND: 1) inhibition of hexokinase II, 2) interference with mitochondrial electron transport, 3) inhibition of cellular lactate export through the monocarboxylate transporters (MCT). We are proposing a fourth mechanism to explain the bioenergetic decline of the tumor following treatment with LND: 4) inhibition of the putative mitochondrial pyruvate carrier (MPC), which would deplete the tumor cells of a key substrate for oxidative phosphorylation and induce an increase in glycolytic metabolism to compensate for decreased oxidative ATP production. We have recently validated a novel extension of isotopomer analysis called cumomer analysis and applied it to perfused DB1 melanoma cells. We believe that this is the first validated metabolic network model of tumor energy metabolism. We propose to test the hypotheses that 1) selective tumor acidification results from inhibition of MCT1, and 2) tumor de-energization is caused by inhibition of the MPC. In Aim 1 of this proposal, we propose to use 13C magnetic resonance spectroscopy (MRS) and liquid chromatography-mass spectrometry (LC-MS) in conjunction with bonded cumomer analysis to test these hypotheses in perfused DB1 and DB8 melanoma cells and in in vivo xenografts of these tumor lines. In Aim 2 we will directly measure the inhibitory effect of LND on MCT1 and MCT4 expressed in Xenopus laevis and will also evaluate the effect of LND on isolated liver and cardiac mitochondria and on permeabilized DB1 and DB8 cells. All of the criticisms of the previous review have been addressed including the critical issue of clinical translation for which we assembled a team of leading experts on melanoma who recommended: 1) define the mechanism of LND (the aim of this proposal), 2) first incorporate LND into hyperthermic isolated limb perfusion of melanoma with melphalan (a method already in clinical practice), and 3) then proceed to systemic therapy of cancers that are already treated with N-mustards or doxorubicin. This project will have a major impact on elucidating the mechanism of activity of a new class of drugs that like LND inhibit MCTs and other key transporters in tumor cells and thereby modify the tumor microenvironment to augment the activity of conventional anticancer agents. It will also develop novel metabolomic methods for the study of tumor metabolism and mechanisms of cancer drug activity.